The proposed research for the next grant period is focused on prokaryotic respiratory oxygen reductases. Oxygenic respiration is responsible for providing virtually all the energy required by aerobic organisms. There are two families of enzymes which catalyze the reduction of O2 to water and conserve the free energy liberated by this reaction by generating a proton motive force- (1) The heme-copper oxygen reductases; (2) the bd-type oxygen reductases. The heme-copper oxygen reductases are themselves subdivided into 3 major subfamilies that are highly divergent- the A-, B- and C-families. In the last grant period we definitively showed that members of each of the 3 families of heme-copper oxygen reductases pump protons. We will pursue two major thrusts in the next grant period. Of the greatest medical relevance is the fact than many human pathogens contain either a C-family oxygen reductase (cytochrome cbb3) and/or cytochrome bd. The reason is that these enzymes are adapted to function best under conditions of low [O2], such as inside macrophages or in granulomas or in the intestine. Under conditions in which O2 is essential for the bioenergetic needs of the pathogen, inhibiting cytochrome cbb3 or cytochrome bd will be lethal. We have designed a high-throughput drug screening protocol that will allow us to identify inhibitors of aerobic respiration in live bacteria. We will focus on 1) Mycobacterium tuberculosis, which requires cytochrome bd in the persistent (dormant) state; 2) Haemophilus influenzae, for which cytochrome bd has been recently shown to be essential for growth; and 3) Neisseria gonorrhoeae, which relies on cytochrome cbb3 for aerobic growth. Respiratory oxygen reductases are a new class of drug targets and we plan to demonstrate their importance in the next grant period. At the same time, we will continue our work on structure/function relationships in the heme-copper oxygen reductases. An important open question is what group or groups are directly involved in proton pumping. We have proposed that the propionate-A group of the active-site heme is the most likely proton loading site of the pump. This will be examined by time-resolved FTIR to directly observe protonation of the proton loading site. We also plan to address the question about how O2 diffuses through the protein to the active site, and how the pathways for this differ in the A-, B- and C-families of oxygen reductases.